The objectives of this investigation are to detect, quantitate and identify free radical metabolites generated from endogenous and exogenous chemicals by peroxidase enzymes. Electron spin resonance (ESR) investigations of the prostaglandin hydroperoxidase and a model enzyme system, horseradish peroxidase, have demonstrated the enzymatic formation of free radical metabolites. The oxidation of benzidine was studied using horesradish peroxidase and prostaglandin synthase. Benzidine was metabolized to a radical cation and a charge-transfer complex composed of the benzidine and its two-electron (di-imine) oxidation product. The di-imine is a resonance structure of the nitrenium ion, the proposed ultimate carcinogenic metabolite of aromatic amines. ESR, on a millisecond time scale, has revealed the formation of a transient phenoxyl radical in the reaction of acetaminophen with horseradish peroxidase/H202 and bovine lactoperoxidase/H202. The short-lived radical is clearly distinguished from the persistent paramagnetic melanin polymers that are generated by prolonged incubation of acetaminophen in the presence of oxidizing enzymes. Ram seminal vesicles and acetaminophen under fast-flow conditions demonstrated the oxidation of acetaminophen to its phenoxyl free radical by the mammalian enzyme prostaglandin hydroperoxidase. Sulfur-centered free radicals have been detected when cysteine was incubated with horseradish peroxidase and H202. In the presence of either molecular oxygen or hydrogen peroxide, the thiyl radical is converted to the cysteine sulfonic and sulfinic acids. Reduced glutathione (GSH) was also oxidized to a sulfur-centered radical (GS.) by horseradish peroxidase and H202. Since cysteine and glutathione play an important role in the structure and function of sulfhydryl-containing proteins, these oxidation reactions may modulate the biological function of these compounds.